This grant will develop a novel strategy to enhance the analytical sensitivity of polymerase chain reaction (PCR)-based detection of bacteria in samples. We will commercialize the strategy in the form of a pre-PCR turbo-charger to be marketed in conjunction with PCR-based molecular diagnostic systems. The current STTR project will apply this strategy to a task for which an ultra-sensitive molecular method is needed, namely the same-day diagnosis of smear-negative and extra-pulmonary tuberculosis (TB) in HIV-coinfected patients. Current molecular TB tests are not sensitive enough for this task. Under ideal conditions PCR can detect a single nucleic acid molecule. However, when applied to real samples its sensitivity is blunted by background nucleic acid and inhibitory substances. To address this limitation, we developed a molecular viability testing (MVT) method that is 5-fold to >10-fold more sensitive than standard PCR when used to detect bacteria in complex samples. The overall Phase 1 + Phase 2 STTR project will develop a semi-automated MVT system for ultra-sensitive detection of Mycobacterium tuberculosis cells in sputum and in cerebrospinal fluid (CSF). Its intended use is the same-day diagnosis of smear-negative pulmonary TB and extrapulmonary TB, which are more commonly seen in HIV-coinfected people than in the general population. Specific aims of this Phase 1 STTR grant are 1 ) to demonstrate a manual MVT assay for M. tuberculosis in sputum and CSF, with an analytical limit of detection 5- to 10-fold better than existing PCR tests; and 2 ) to validate an inexpensive single-use blender system for efficient release of target RNA from M. tuberculosis cells in sputum and CSF. In Phase 2 of the project, MVT will be automated to yield a single- step pre-PCR unit that will be commercialized in conjunction with existing qPCR system(s).